An infrared and circular dichroism combined approach to the analysis of protein secondary structure.

Selected regions of infarred (ir) and circular dichroism (CD) spectral data from 10 proteins were combined and analyzed by a factor analysis method. The regions consisted of the area normalized amide I region from 1700 to 1600 cm-1 for the ir spectra and from 178 to 240 nm for the CD spectra. Each CD spectrum was scaled by a factor of 0.5 before appending the data to the ir spectral data. The scaling factor was deemed necessary to account for relative intensity differences between the ir and CD data and provided nearly optimum agreement between secondary structure estimated by the combined approach to secondary structure determined by X-ray crystallography. The ir/CD combined approach to estimation of helix, beta-sheet, beta-turn, and other or undefined secondary structure agreed with X-ray crystallographic determined structure better than estimation using data from either method alone. Correlation coefficients between X-ray and ir/CD combined secondary structure determinations were 0.99 for helix, 0.90 for beta-sheet, 0.70 for beta-turn, and 0.78 for other structure. The four most significant eigenvectors or basis spectra from eigenanalysis of the ir/CD data are presented as well as generalized inverse spectra for four secondary structures.     

External reflection FTIR of peptide monolayer films in situ at the air/water interface: experimental design, spectra-structure correlations, and effects of hydrogen-deuterium exchange.

A Fourier transform infrared spectrometer has been interfaced with a surface balance and a new external reflection infrared sampling accessory, which permits the acquisition of spectra from protein monolayers in situ at the air/water interface. The accessory, a sample shuttle that permits the collection of spectra in alternating fashion from sample and background troughs, reduces interference from water vapor rotation-vibration bands in the amide I and amide II regions of protein spectra (1520-1690 cm-1) by nearly an order of magnitude. Residual interference from water vapor absorbance ranges from 50 to 200 microabsorbance units. The performance of the device is demonstrated through spectra of synthetic peptides designed to adopt alpha-helical, antiparallel beta-sheet, mixed beta-sheet/beta-turn, and unordered conformations at the air/water interface. The extent of exchange on the surface can be monitored from the relative intensities of the amide II and amide I modes. Hydrogen-deuterium exchange may lower the amide I frequency by as much as 11-12 cm-1 for helical secondary structures. This shifts the vibrational mode into a region normally associated with unordered structures and leads to uncertainties in the application of algorithms commonly used for determination of secondary structure from amide I contours of proteins in D2O solution.     

Determination of secondary structure of globular proteins using circular dichroism spectra

A ridge regression method is presented for prediction of the secondary structure of proteins by the circular dichroism spectra (CD) from 190 to 236 nm. Eight types of the secondary structure were calculated on a microcalculator. The method is based on the X-ray data of Kabsh and Sander. The teaching rule is constructed on CD spectra of 30 proteins of all structural classes of the globular proteins (alpha, alpha/beta, alpha + beta and beta-proteins). The errors of the methods are analysed by removing each protein from the reference set and analyzing its structure in terms of the remaining proteins. Correlation coefficients and root-mean square deviations between CD and X-ray data were: 0.99 and 0.03 for alpha-helix, 0.86 and 0.02 for 3(10)-helix, 0.92 and 0.06 for antiparallel beta-sheet, 0.86 and 0.03 for parallel beta-sheet, 0.94 and 0.01 for T3 beta-turn, 0.85 and 0.02 for other beta-turn, 0.84 and 0.03 for S-bends, 0.83 and 0.04 for "random" structure.     

Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication

Inverse circular dichroism (CD) spectra are presented for each of the five major secondary structures of proteins: alpha-helix, antiparallel and parallel beta-sheet, beta-turn, and other (random) structures. The fraction of the each secondary structure in a protein is predicted by forming the dot product of the corresponding inverse CD spectrum, expressed as a vector, with the CD spectrum of the protein digitized in the same way. We show how this method is based on the construction of the generalized inverse from the singular value decomposition of a set of CD spectra corresponding to proteins whose secondary structures are known from X-ray crystallography. These inverse spectra compute secondary structure directly from protein CD spectra without resorting to least-squares fitting and standard matrix inversion techniques. In addition, spectra corresponding to the individual secondary structures, analogous to the CD spectra of synthetic polypeptides, are generated from the five most significant CD eigenvectors.     

Determination of the secondary structure content of proteins in aqueous solutions from their amide I and amide II infrared bands. Comparison between classical and partial least-squares methods

A method for estimating protein secondary structure from infrared spectra has been developed. The infrared spectra of H2O solutions of 13 proteins of known crystal structure have been recorded and corrected for the spectral contribution of water in the amide I and II region by using the algorithm of Dousseau et al. [Dousseau, F., Therrien, M., & Pézolet, M. (1989) Appl. Spectrosc. 43, 538-542]. This calibration set of proteins has been analyzed by using either a classical least-squares (CLS) method or the partial least-squares (PLS) method. The pure-structure spectra calculated by the classical least-squares method are in good agreement with spectra of poly(L-lysine) in the alpha-helix, beta-sheet, and undefined conformations. The results show that the best agreement between the secondary structure determined by X-ray crystallography and that predicted by infrared spectroscopy is obtained when both the amide I and II bands are used to generate the calibration set, when the PLS method is used, and when it is assumed that the secondary structure of proteins is composed of only four types of structure: ordered and disordered alpha-helices, beta-sheet, and undefined conformation. Attempts to include turns in the secondary structure estimation have led to a loss of accuracy. The standard deviation of the difference between X-ray and infrared secondary structure estimates with this method is 4.8% for the alpha-helix, 3.7% for the beta-sheet, and 5.1% for the undefined structure, whereas the regression coefficients are 0.95, 0.96, and 0.56, respectively. The spectra of the calibration proteins were also recorded in 2H2O solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of protein far UV circular dichroism spectra by neural networks.

A new method based on neural network theory is presented to analyze and quantify the information content of far UV circular dichroism spectra. Using a backpropagation network model with a single hidden layer between input and output, it was possible to deduce five different secondary structure fractions (helix, parallel and antiparallel beta-sheet, beta-turn and random coil) with satisfactory correlations between calculated and measured secondary structure data. We demonstrate that for each wavelength interval a specific network is suitable. The remaining discrepancy between the secondary structure data from neural network prediction and crystallography may be attributed to errors in the determination of protein concentration and random noise in the CD signal, as indicated by simulations.     

Estimation of protein secondary structure and error analysis from circular dichroism spectra

The estimation of protein secondary structure from circular dichroism spectra is described by a multivariate linear model with noise (Gauss-Markoff model). With this formalism the adequacy of the linear model is investigated, paying special attention to the estimation of the error in the secondary structure estimates. It is shown that the linear model is only adequate for the alpha-helix class. Since the failure of the linear model is most likely due to nonlinear effects, a locally linearized model is introduced. This model is combined with the selection of the estimate whose fractions of secondary structure summate to approximately one. Comparing the estimation from the CD spectra with the X-ray data (by using the data set of W.C. Johnson Jr., 1988, Annu. Rev. Biophys. Chem. 17, 145-166) the root mean square residuals are 0.09 (alpha-helix), 0.12 (anti-parallel beta-sheet), 0.08 (parallel beta-sheet), 0.07 (beta-turn), and 0.09 (other). These residuals are somewhat larger than the errors estimated from the locally linearized model. In addition to alpha-helix, in this model the beta-turn and "other" class are estimated adequately. But the estimation of the antiparallel and parallel beta-sheet class remains unsatisfactory. We compared the linear model and the locally linearized model with two other methods (S. W. Provencher and J. Gl?ckner, 1981, Biochemistry 20, 1085-1094; P. Manavalan and W. C. Johnson Jr., 1988, Anal. Biochem. 167, 76-85). The locally linearized model and the Provencher and Gl?ckner method provided the smallest residuals. However, an advantage of the locally linearized model is the estimation of the error in the secondary structure estimates.     

Structural composition of betaI- and betaII-proteins

Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of alpha-rich proteins are similar to those of model alpha-helices, but beta-rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model beta-sheets or unordered polypeptides. The existence of these two types of CD spectra for beta-rich proteins form the basis for their classification as betaI- and betaII-proteins. Although the conformation of beta-sheets is largely responsible for the CD spectra of betaI-proteins, the source of betaII-protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II-type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the beta-sheet and P2 structure contents in beta-rich proteins to understand the origin of betaII-protein CD. We find that betaII-proteins have a ratio of P2 to beta-sheet content greater than 0.4, whereas for betaI-proteins this ratio is less than 0.4. The beta-sheet content in betaI-proteins is generally higher than that in betaII-proteins. The origin of two classes of CD spectra for beta-rich proteins appears to lie in their relative beta-sheet and P2 structure contents.     

Conformation similarities of ricin A-chain and trichosanthin

The conformation of ricin A-chain from castor bean was studied by circular dichroism at pH 4.7, 7 and 9 and compared with that of trichosanthin from the Chinese herb Tianhuafen. The CD spectra of ricin A-chain and trichosanthin were nearly identical at each of the three pHs. Analysis of the data indicated that, like trichosanthin, ricin A-chain had about 29% alpha-helix and 42% beta-sheet but no beta-turn. However, there was a subtle difference in the CD spectra in 20 mM sodium dodecyl sulfate, the addition of which at pH 7 slightly increased the helicity and decreased the content of beta-sheet of ricin A-chain in contrast to a larger increase in helicity at the expense of beta-sheet for trichosanthin, thus indicating a different stability against the surfactant. Native ricin A-chain and trichosanthin had about the same amount of secondary structure, which supports the belief that a high degree of sequence homology of the two proteins [Zhang & Wang (1986) Nature 321, 477-478] may lead to a conformational similarity between them, even though the two proteins are not taxonomically related.     

Evaluation of the information content in infrared spectra for protein secondary structure determination

Fourier-transform infrared spectroscopy is a method of choice for the experimental determination of protein secondary structure. Numerous approaches have been developed during the past 15 years. A critical parameter that has not been taken into account systematically is the selection of the wavenumbers used for building the mathematical models used for structure prediction. The high quality of the current Fourier-transform infrared spectrometers makes the absorbance at every single wavenumber a valid and almost noiseless type of information. We address here the question of the amount of independent information present in the infrared spectra of proteins for the prediction of the different secondary structure contents. It appears that, at most, the absorbance at three distinct frequencies of the spectra contain all the nonredundant information that can be related to one secondary structure content. The ascending stepwise method proposed here identifies the relevance of each wavenumber of the infrared spectrum for the prediction of a given secondary structure and yields a particularly simple method for computing the secondary structure content. Using the 50-protein database built beforehand to contain as little fold redundancy as possible, the standard error of prediction in cross-validation is 5.5% for the alpha-helix, 6.6% for the beta-sheet, and 3.4% for the beta-turn.     

Secondary structure of the mammalian 70-kilodalton heat shock cognate protein analyzed by circular dichroism spectroscopy and secondary structure prediction

Heat shock proteins are rapidly synthesized when cells are exposed to stressful agents that cause protein damage. The 70-kDa heat shock induced proteins and their closely related constitutively expressed cognate proteins bind to unfolded and aberrant polypeptides and to hydrophilic peptides. The structural features of the 70-kDa heat shock proteins that confer the ability to associate with diverse polypeptides are unknown. In this study, we have used circular dichroism (CD) spectroscopy and secondary structure prediction to analyze the secondary structure of the mammalian 70-kDa heat shock cognate protein (hsc 70). The far-ultraviolet CD spectrum of hsc 70 indicates a large fraction of alpha-helix in the protein and resembles the spectra one obtains from proteins of the alpha/beta structural class. Analysis of the CD spectra with deconvolution methods yielded estimates of secondary structure content. The results indicate about 40% alpha-helix and 20% aperiodic structure within hsc 70 and between 16-41% beta-sheet and 21-0% beta-turn. The Garnier-Osguthorpe-Robson method of secondary structure prediction was applied to the rat hsc 70 amino acid sequence. The predicted estimates of alpha-helix and aperiodic structure closely matched the values derived from the CD analysis, whereas the predicted estimates of beta-sheet and beta-turn were midway between the CD-derived values. Present evidence suggests that the polypeptide ligand binding domain of the 70-kDa heat shock protein resides within the C-terminal 160 amino acids [Milarski, K. L., & Morimoto, R. I. (1989) J. Cell Biol. 109, 1947-1962].(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox-dependent changes in beta-extended chain and turn structures of cytochrome c in water solution determined by second derivative amide I infrared spectra.

The redox-dependent changes in secondary structure of cytochromes c from horse, cow, and dog hearts in water at 20 degrees C have been determined by amide I infrared spectroscopy. Second derivative amide I spectra were obtained by use of a procedure that includes a convenient method for the effective subtraction of the spectrum of water vapor in the system. The band at 1657 cm-1 representing the helix structure was unaffected by a change in redox state whereas changes in bands due to turns at 1680, 1672, and 1666 cm-1, unordered structure at 1650 cm-1, and beta-structures at 1632 and 1627 cm-1 occurred. About one-fourth of the beta-extended chain spectral region and one-fifth of the beta-turn region (involving a total of approximately 9-13 residues) were sensitive to the oxidation state of heme iron. No significant changes in the secondary structure of either the reduced or oxidized protein due to changes in ionic strength were detected. The localized structural rearrangements triggered by the changes in oxidation state of heme iron are consistent with differences in the binding of heme iron to a histidine imidazole nitrogen and a methionine sulfur atom from the beta-extended chain. The demonstrated ability to obtain highly reproducible second derivative amide I infrared spectra confirms the unique utility of such spectral measurements for localization of subtle changes in secondary structure within a protein, especially for changes among the multiple turns and beta-structures.     

Raman spectroscopic study of mechanically deformed poly-L-alanine

Raman spectroscopy has been used in investigating the conformational transitions of poly-L -alanine (PLA) induced by mechanical deformation. We see evidence of the alpha-helical, antiparallel beta-sheet, and a disordered conformation in PLA. The disordered conformation has not been discussed in previous infrared and X-ray diffraction investigations and may have local order similar to the left-handed 3₁ poly glycine helix. The amide III mode in the Raman spectrum of PLA is more sensitive than the amide I and II modes to changes in secondary structure of the polypeptide chain. Several lines below 1200 cm^?1 are conformationally sensitive and may generally be useful in the analysis of Raman spectra of proteins. A line at 909 cm^?1 decreases in intensity after deformation of PLA. In general only weak scattering is observed around 900 cm^?1 in the Raman spectra of antiparallel beta-sheet polypeptides. The Raman spectra of the amide N–H deuterated PLA and poly-L -leucine (PLL) in the alpha-helical conformation and poly-L -valine (PLV) in the beta-sheet conformation are presented. Splitting is observed in the amide III mode of PLV and the components of this mode are assigned. The Raman spectrum of an alpha-helical random copolymer of L -leucine and L -glutamic acid is shown to be consistent with the spectra of other alphahelical polypeptides.     

The lysine and methionine rich basic subunit of buckwheat grain legumin: some results of a structural study.

The 26 kD basic subunit of 280 kD buckwheat grain legumin has been partially characterized by measurement of its fluorescence and CD spectra. The protein has 22% alpha-helix, 36% beta-sheet, 12% beta-turn and 30% random coil secondary structure. In comparison with the basic subunits of other legumin-type proteins, the buckwheat legumin subunit has a high content of lysine and methionine. The protein also has higher ratios of lysine to arginine and methionine to arginine.     

Conformational change of chaperone Hsc70 upon binding to a decapeptide: a circular dichroism study.

The conformation of bovine Hsc70, a 70-kDa heat shock cognate protein, and its conformational change upon binding to decapeptides, was studied by CD spectroscopy and secondary structure prediction (Chou, P.Y. & Fasman, G.D., 1974, Biochemistry 13, 222-245). The CD spectra were analyzed by the LINCOMB method, as well as by the convex constraint analysis (CCA) method (Perczel, A., Park, K., & Fasman, G.D., 1992, Anal. Biochem. 203, 83-93). The result of the CD analysis of Hsc70 (15% alpha-helix, 24% beta-sheet, 24% beta-turn, and 38% remainder) was very similar to the predicted secondary structure for the beta-sheet (24%) and the beta-turn (29%). However, there is disagreement between the alpha-helical content by CD analysis (15%) and the predicted structure (30%). In spite of the fact that the decapeptides contained a considerable amount of beta-sheet (22%), the interaction of the heat shock protein with the peptide resulted in an overall decrease in the content of beta-sheet conformation (-15%) of the complex. This may be due to induction of a molten globule state. The result of the CCA analysis indicated that the Hsc70 undergoes a conformational change upon binding the decapeptides.     

A holistic approach for protein secondary structure estimation from infrared spectra in H(2)O solutions

We present an improved technique for estimating protein secondary structure content from amide I and amide III band infrared spectra. This technique combines the superposition of reference spectra of pure secondary structure elements with simultaneous aromatic side chain, water vapor, and solvent background subtraction. Previous attempts to generate structural reference spectra from a basis set of reference protein spectra have had limited success because of inaccuracies arising from sequential background subtractions and spectral normalization, arbitrary spectral band truncation, and attempted resolution of spectroscopically degenerate structure classes. We eliminated these inaccuracies by defining a single mathematical function for protein spectra, permitting all subtractions, normalizations, and amide band deconvolution steps to be performed simultaneously using a single optimization algorithm. This approach circumvents many of the problems associated with the sequential nature of previous methods, especially with regard to removing the subjectivity involved in each processing step. A key element of this technique was the calculation of reference spectra for ordered helix, unordered helix, sheet, turns, and unordered structures from a basis set of spectra of well-characterized proteins. Structural reference spectra were generated in the amide I and amide III bands, both of which have been shown to be sensitive to protein secondary structure content. We accurately account for overlaps between amide and nonamide regions and allow different structure types to have different extinction coefficients. The agreement between our structure estimates, for proteins both inside and outside the basis set, and the corresponding determinations from X-ray crystallography is good.     

Secondary structure in properdin of the complement cascade and related proteins: a study by Fourier transform infrared spectroscopy

Six structural repeat motifs of 58 amino acids are found in the sequence of both mouse and human properdins. Twelve more examples of the motif are available from the sequences of thrombospondin, the terminal complement components, and the thrombospondin-related anonymous protein. The averaged Robson and Chou-Fasman secondary structure predictions show that there are 57-66% turn and 19-38% beta-sheet structures in the typical repeat motif. The high amount of turn structure is consistent with Gly, Pro, Cys, and Ser being the four most abundant amino acid residues in properdin. Comparisons with sequences found in the circumsporozoite protein from several species of malaria parasites show that their sequences and secondary structures strongly coincide only in a 18-residue segment. Further secondary structure analysis utilized Fourier transform infrared spectroscopy of human properdin in 2H2O buffers. These show a broad amide I band that, after second-derivative and deconvolution calculations, is shown to be composed of several components. Two at 1633 and 1683 cm-1 are strong evidence for beta-sheet structure, although overlap from beta-turns can also contribute. The presence of beta-turn structure is indicated by absorptions at 1662-1675 and 1645 cm-1. The properdin structure contains substantial quantities of beta-sheet and beta-turn structures, which is consistent with the secondary structure predictions and amino acid compositions. The length of the repeat motif is estimated as 3.3-4.3 nm, and an estimated 14-22% of nonexchanged amide protons reside in properdin. This is suggestive of a high degree of solvent accessibility in the structure.     

Protein secondary structures in water from second-derivative amide I infrared spectra

Infrared spectra have been obtained for 12 globular proteins in aqueous solution at 20 degrees C. The proteins studied, which vary widely in the relative amounts of different secondary structures present, include myoglobin, hemoglobin, immunoglobulin G, concanavalin A, lysozyme, cytochrome c, alpha-chymotrypsin, trypsin, ribonuclease A, alcohol dehydrogenase, beta 2-microglobulin, and human class I major histocompatibility complex antigen A2. Criteria for evaluating how successfully the spectra due to liquid and gaseous water are subtracted from the observed spectrum in the amide I region were developed. Comparisons of second-derivative amide I spectra with available crystal structure data provide both qualitative and quantitative support for assignments of infrared bands to secondary structures. Band frequency assignments assigned to alpha-helix, beta-sheet, unordered, and turn structures are highly consistent among all proteins and agree closely with predictions from theory. alpha-Helix and unordered structures can each be assigned to only one band whereas multiple bands are associated with beta-sheets and turns. These findings demonstrate a method of analysis of second-derivative amide I spectra whereby the frequencies of bands due to different secondary structures can be obtained. Furthermore, the band intensities obtained provide a useful method for estimating the relative amounts of different structures.     

Synthesis and spectroscopy of membrane receptor proteins. The gamma subunit of the IgE receptor.

The high-affinity receptor for IgE is a tetrameric complex of subunits of the type alpha beta gamma 2. We report here conformational studies of the intact gamma subunit in trifluoroethanol and water/liposomes by circular dichroism and Fourier-transform infrared (FTIR) spectroscopy. In trifluoroethanol, the FTIR amide I' frequencies were consistent with two predominant conformational components, the beta-turn and alpha-helix, whilst in liposomes consisting of D2O and dimyristoylglycerophosphocholine (Myr2GroPCho), three components were observed. The third component present may contain some left-handed extended helix. Spectral simulation was carried out to demonstrate that the CD spectra were consistent with the component conformations identified from FTIR spectroscopy. The stimulated CD spectra were in excellent agreement with the experimental spectra. The intact gamma subunit conformation in trifluoroethanol was shown to possess 72% alpha-helical and 28% beta-turn conformations. In water/Myr2GroPCho liposomes the percentage of each conformational component present is 37%, 38% and 25% for the alpha-helix, beta-turn and extended structures, respectively. Assuming that the transmembrane fragment was alpha-helical, an excellent correlation was found between this derived alpha-helical content in water/liposomes (37%) and from hydrophobicity plots where the percentage of amino acids in the transmembrane domain is predicted by others to be 34%. It is suggested that the beta-turn detected by CD and FTIR was attributable to a 3(10) helix rather than a type I or type III reverse turn.     

Secondary structure of a core protein from pig skin proteodermatan sulfate: CD and Fourier transform IR spectroscopic studies in solution

The secondary structure of a 38 kDa core protein from pig skin proteodermatan sulfate (PDS), was investigated in solution using CD and Fourier transform (FT) ir spectroscopy. Both techniques generally have provided complementary data on the secondary structures of proteins. CD spectral analysis has shown that the core protein contains 60% beta-turn and alpha-helical structures, the rest being "unordered" structure. FT ir data do not permit calculation of quantitative contributions of substructures, at the present time, to the overall secondary structure of the core protein. CD spectrum of the intact PDS is similar to the core protein CD spectrum.